This work was supported by grants from the National Institutes of Health to V.F. (NS42599 and GM077569).

1. Preparing for Crosslinking
<ol>
	<li>You will need to plate a sufficient number of cells to allow isolation of 500 &mu;g of protein per standard tube reaction. A single tube may be enough for identification of putative interactors of a protein of interest by immunoblot. In this case bead bound material can be eluted with SDS-PAGE sample buffer (immunoprecipitation). For mass spectrometry analysis, the number of standard reactions should be increased at least ten times and protein complexes should be eluted by out-competition with a peptide antigen recognized by the antibody used (immunoaffinity chromatography). This strategy will allow the isolation of protein complexes free of immunoglobulin.</li>
	<li>The optimal cell density for crosslinking is between 75% and 90% confluency. In high confluency conditions, cells tend to pile on each other, which decreases accessibility to the cell-permeable crosslinker to all cells.</li>
	<li>Cell type and experimental condition should be labeled on the bottom of the cell plate. We routinely include controls with vehicle alone.</li>
	<li>Prepare all solutions EXCEPT the DSP solution prior to crosslinking.
 <ol>
		<li>Prepare a stock of phosphate-buffered saline buffer with CaCl2 0.1 mM and CaCl2 1 mM (PBS/Ca/Mg) prior to starting the experiment. The addition of calcium and magnesium ions is critical for adhesion of cells to the culture plate during the course of the experiment. Store at 4&deg;C.</li>
		<li>50X Complete Protease Inhibitor Cocktail - 1 tablet dissolved in 1mL Milli-Q water. Store at -20&deg;C.</li>
		<li>20% Triton X-100 Weigh out 10g Triton X -100 and dilute in total volume of 50 ml Milli-Q Water. Rock at 4&deg;C overnight and store at 4&deg;C until use. Do not store for more than 1 month. Use dilutions of this 20% stock to prepare the lysis and IP buffers described below.</li>
		<li>50X DSP Quenching Solution 1M TRIS to pH 7.4. Store at room temperature.</li>
		<li>Prepare a 10x Buffer A solution. 
		10X Buffer A:
 <ul>
			<li>50 mL 1 M HEPES</li>
			<li>150 mL 5 M NaCl</li>
			<li>10 mL 0.5 M EGTA</li>
			<li>250 &mu;L 2 M MgCl2</li>
			<li>Adjust pH to 7.4</li>
			<li>Bring final volume to 500 mL</li>
 </ul>
The 10X Buffer A solution is diluted to 1X Buffer A as needed. 1x Buffer A is also used in lysate and immuno-magnetic-precipitation buffers. 
	Buffer A 1X
		<ul>
			<li>10 mM Hepes</li>
			<li>150 mM NaCl</li>
			<li>1 mM EGTA</li>
			<li>0.1 mM MgCl2</li>
			<li>pH 7.4</li>
 </ul>
 </li>
	<li>Lysis Buffer 1X Buffer A + 0.5% Triton X-100.</li>
	<li>Immuno-Magnetic Precipitation Buffer (IP Buffer) 1X Buffer A + 0.1% Triton X-100.</li>
	</ol>
</li>
</ol>
2. Preparing the Crosslinking Solution
<ol>
	<li>Prepare the DSP solution immediately before applying to cells. DSP is highly hydrophobic and should be dissolved in DMSO before diluting into PBS/Ca/Mg buffer. Dissolve 40 mg of DSP in 1 mL of DMSO. This makes a 100 mM solution of DSP (The molecular weight of DSP is 404.42 g/mol).</li>
	<li>Warm an appropriate volume of PBS/Ca/Mg to 37&deg;C in order to facilitate dilution of DSP/DMSO into PBS. 
Volumes needed for assorted plate sizes: 6-well plate 2 mL per well 10 cm plate 10 mL per plate 15 cm plate 20 mL per plate</li>
	<li>Add 10&mu;L of DSP/DMSO stock solution for every 1 mL of warm PBS/Ca/Mg. Add DSP/DMSO stock solution drop by drop with repeated mixing until all the DSP has dissolved. Make a control solution of 10 &mu;L DMSO added to every 1 mL of PBS/Ca/Mg.</li>
	<li>Place all PBS/Ca/Mg solutions in an ice-water bath no longer than 10 min.</li>
</ol>
3. Prepare Cells for Crosslinking
<ol>
	<li>Prepare an ice-water bath that will fit all plates for crosslinking or vehicle control incubation.</li>
	<li>Take plates from the 37&deg;C incubator and place them immediately into the ice-water bath.</li>
	<li>Wash cells two times with ice-cold PBS/Ca/Mg. Use the same volume that you will use for the crosslinker incubation (see section 2.2).</li>
	<li>Remove second wash and add either vehicle control or DSP crosslinking buffer solution.</li>
	<li>Incubate on ice for two hours. You should check the plates approximately every twenty minutes to ensure that all cells are covered by solution. You may notice a small amount of DSP precipitate out of solution. This is normal.</li>
</ol>
4. Inactivation of DSP Reaction
<ol>
	<li>Prepare an 1X DSP quenching solution of 20 mM Tris pH 7.4 in PBS/Ca/Mg (20 &mu;L of 1 M Tris pH 7.4 for every 1 mL of PBS/Ca/Mg).</li>
	<li>Remove the vehicle control and crosslinking solutions. </li>
	<li>Add ice-cold inactivation solution and incubate on ice for 15 minutes.</li>
</ol>
5. Cell Lysis
<ol>
	<li>During DSP quenching incubation, prepare the cell lysis buffer of 0.5% Triton X-100 in Buffer A with Complete protease inhibitor cocktail. Add 20 &mu;L 50X stock solution of Complete for every 1 mL lysis buffer.</li>
	<li>Following the 15 minute DSP quenching incubation, wash cells two times with PBS/Ca/Mg.</li>
	<li>Add lysis buffer + Complete to cells and rock at 4&deg;C for 30 minutes. Some suggested volumes of lysis buffer for assorted plate sizes:
 <ul>
 	<li>6-well plate 0.5 mL per well</li>
 	<li>10 cm plate 1 mL per plate</li>
 	<li>15 cm plate 1 mL per plate</li>
 </ul>
 </li>
	<li>After lysis, use a cell scraper to collect the cells from the plate. Be sure to keep lysates on ice to minimize protease activity</li>
	<li>Spin cell lysates for 15 minutes at 4&deg;C in a bench-top mini-centrifuge at top speed (15.000 x g). </li>
	<li>Pipette the supernatant into a fresh tube. Discard the pellet.</li>
	<li>Analyze protein levels and proceed with immunoprecipitation.</li>
 </ol>
6. Preparing Immuno-magnetic Precipitation Beads
Note: This step is usually started directly after beginning the 2 hour crosslinking incubation.
<ol>
	<li>Combine 30 &mu;L of Dynal immunomagnetic beads, 500 &mu;L of IP Buffer (1x Buffer A + 0.1% Triton X-100) and the appropriate amount of antigen-specific antibody to screwtop microcentrifuge tubes. Prepare antibody-free and non-specific antibody tubes for negative controls. The appropriate amount of antibody should be determined based on individual antigens during separate experiments. Label all IP tubes.</li>
	<li>Allow the IP tubes to incubate in an end-over-end rocker at room temperature for two hours. Alternatively, incubate the IP tubes with antibody overnight at 4&deg; the day prior to crosslinking.</li>
	<li>After the incubation, do a quick 10 second mini-centrifugation to collect the beads at the bottom of the tube.</li>
	<li>Slide the tubes into the magnetic holder, which will pull the magnetic beads away from the bottom of the tube. With the beads safely out of the way, you can now easily wash away any unbound antibody.</li>
	<li>Use a small bore aspirator tip such as a gel loading tip, or p200 tip to remove the incubation buffer and any unbound antibody. As soon as the incubation volume is removed add in 1 mL of IP Buffer.</li>
	<li>Cap the tube, gently re-suspend the beads, and incubate all IP tubes at room temperature for 5 minutes with end-over-end rotation.</li>
	<li>Repeat the 5 minute wash step (steps 6.3 thru 6.6) once more with a fresh milliliter of IP Buffer.</li>
	<li>The IP tubes can remain in the last wash incubation until the crosslinked lysate is ready to be added to the beads.</li>
</ol>
7. Incubate Crosslinked Lysate with Immuno-magnetic Beads
<ol>
	<li>After the last wash spin immuno-magnetic beads, down in a mini-centrifuge for 10 seconds. Then proceed to slip the tubes into the magnetic holder.</li>
	<li>Again, using a small bore aspiration tip, remove the incubation volume from the tube. Immediately add 500 &mu;L of crosslinked cell lysate (assuming lysate at 1 &mu;g/mL) to the tubes containing immuno-magnetic beads. If you will be using peptide competition as a negative control, an appropriate amount of peptide should be included at this point. Previous experiments should be performed to determine appropriate peptide competition conditions. Alternatively, you may want to have lysate free immuno-magnetic beads as a negative control. In this case, immediately add 500 &mu;L of Lysate Buffer (1x Buffer A + 0.5% Triton X-100) + Complete to the immuno-magnetic beads.</li>
	<li>Gently resuspend the beads. Resuspended beads should be incubated at 4&deg; in an end-over-end rotation for 2 hours.</li>
</ol>
8. Wash Unbound Lysate from Beads
<ol>
	<li>Once lysate has had sufficient incubation time, do a quick 10 second spin in a mini-centrifuge. Then slide the tubes into the magnetic holder. The magnetic holder should be kept in an ice bath for the remainder of the experiment.</li>
	<li>Using a small bore aspirator tip, remove all unbound lysate. Immediately after all unbound lysate is removed add 1 mL of IP Buffer.</li>
	<li>Cap the tube and gently resuspend the beads. </li>
	<li>Once the beads are resuspended repeat the bead pelleting step (8.1).</li>
	<li>Again aspirate off all of the incubation volume and immediately add 1 mL of IP Buffer, then cap the tube.</li>
	<li>Gently resuspend the beads in IP Buffer. Incubate resuspended beads for 5 minutes at 4&deg; with end-over-end rocking.</li>
	<li>Repeat the bead washing steps (8.4 through 8.6) 4 more times.</li>
</ol>
9. Denature Sample and Collect from Beads
<ol>
	<li>After all of the immuno-magnetic precipitation tubes have been washed, re-sediment the beads (8.1) and slide tubes into the magnetic holder.</li>
	<li>Proteins bound to the immuno-magnetic beads are removed in denaturing conditions. Aspirate off the last IP Buffer wash and remove the IP tube from the magnetic holder.</li>
	<li>Add 1x Gel Sample Buffer to the IP tube just above the bead pellet to pool the beads at the bottom of the tube again. The amount of sample buffer depends on the well size of the gel to be used. You may need to gently tap the bottom of the tube to get all of the beads resuspended in the Gel Sample Buffer.</li>
	<li>Repeat the denaturing process (9.2 through 9.3) for each IP tube.</li>
	<li>Immuno-magnetic beads resuspended in Gel Sample Buffer are then heated at 75&deg;C for 5 minutes to complete the denaturing process.</li>
	<li>Once the sample has been denatured it can be run on an SDS-PAGE gel for western blotting. Emptied Immuno-Magnetic Beads can be re-pelleted by centrifugation and removed from the sample with the magnetic holder.</li>
</ol>
10. Elution of Crosslinked Complexes with Antigenic Peptides (Immunoaffinity Chromatography).
<ol>
	<li>After all of the immuno-magnetic immunoprecipitation tubes have been washed, re-sediment the beads (8.1) and slide tubes into the magnetic holder. Aspirate supernatant and add 10 &mu;l of Buffer A supplemented with the antigenic peptide recognized by the antibody used for immunoisolation of protein complexes. You should test concentrations ranging from 10-200 &mu;M for efficient elution.</li>
	<li>Incubate the beads for 2 h at 4&deg;C.</li>
	<li>Put beads in the magnetic stand and carefully recover the 10 mL of eluted material. Save this supernatant.</li>
	<li>Quickly wash the beads with Buffer A and discard the wash. Save the beads.</li>
	<li>Add Gel Sample Buffer to the saved supernatant and beads to a concentration of 1X. Incubate these tubes at 75&deg;C for 5 minutes.</li>
	<li>Analyze beads and peptide eluate by SDS-PAGE. The eluate should be free of IgG both by protein stain of SDS-PAGE or by immunoblots (Figure 1B). This material free of IgG is suitable for mass spectrometry.</li>
</ol>
<img src="/files/ftp_upload/1855/1855fig1.jpg" alt="Figure 1" /> Figure 1. Isolation of AP-3 interacting protein complexes and membrane proteins. HEK293 cells (A) or PC12 cells (B) were treated either in the presence of vehicle control (DMSO, odd lanes Fig 1A) or DSP (even lanes Fig. 1A or all lanes in Fig 1B). Clarified extracts were incubated either with beads alone (Fig. 1A, Lanes 3-4), transferrin receptor antibodies (Fig. 1A, Lanes 5-6), or AP-3 &delta; antibodies (Fig. 1A, Lanes 7-10; Fig. 1B, lanes 1-10). Immune complexes were eluted with SDS-PAGE sample buffer (Fig. 1A), Buffer A alone or (Fig. 1B, lanes 1-2), or Buffer A supplemented with increasing concentrations of the &delta; antigenic peptide corresponding to the amino acids 680 710 of human &delta;-adaptin (NCBI:AAD03777; gi:1923266)(Fig. 1B, lanes 3-10). This peptide binds the &delta; antibody. After peptide elution, supernatants (S) and beads (B) were analyzed by immunoblot (Fig. 1B). AP-3, which is detected with antibodies against the &delta;, &beta;3, and &sigma;3 subunits, coprecipitates with the following soluble factors: clathrin heavy chain (CHC), the BLOC-1 subunit pallidin, and the HOPS complex subunit vps33b; as well as the membrane proteins phosphatidylinositol-4-kinase type II alpha (PI4KII&alpha;) and the zinc transporter 3 (ZnT3). Note the absence of IgG mouse heavy chains in the peptide eluted supernatant in Fig. 1B.

<ol>
	<li>Salazar, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hermansky-Pudlak+syndrome+protein+complexes+associate+with+phosphatidylinositol+4-kinase+type+II+alpha+in+neuronal+and+non-neuronal+cells.">Hermansky-Pudlak syndrome protein complexes associate with phosphatidylinositol 4-kinase type II alpha in neuronal and non-neuronal cells.</a> J Biol Chem. 284, 1790-1802 (2009).</li><li>Lomant, A. J., Fairbanks, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemical+probes+of+extended+biological+structures:+synthesis+and+properties+of+the+cleavable+protein+cross-linking+reagent+[35S]dithiobis(succinimidyl+propionate.">Chemical probes of extended biological structures: synthesis and properties of the cleavable protein cross-linking reagent [35S]dithiobis(succinimidyl propionate.</a> J Mol Biol. 104, 243-261 (1976).</li><li>Xiang, C. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Using+DSP,+a+reversible+cross-linker,+to+fix+tissue+sections+for+immunostaining,+microdissection+and+expression+profiling.">Using DSP, a reversible cross-linker, to fix tissue sections for immunostaining, microdissection and expression profiling.</a> Nucleic Acids Res. 32, e185-e185 (2004).</li><li>Craige, B., Salazar, G., Faundez, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phosphatidylinositol-4-Kinase+Type+II+Alpha+Contains+an+AP-3+Sorting+Motif+and+a+Kinase+Domain+that+are+both+Required+for+Endosome+Traffic.">Phosphatidylinositol-4-Kinase Type II Alpha Contains an AP-3 Sorting Motif and a Kinase Domain that are both Required for Endosome Traffic.</a> Mol Biol Cell. 19, 1415-1426 (2008).</li><li>Newell-Litwa, K., Seong, E., Burmeister, M., Faundez, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuronal+and+non-neuronal+functions+of+the+AP-3+sorting+machinery.">Neuronal and non-neuronal functions of the AP-3 sorting machinery.</a> J Cell Sci. 120, 531-541 (2007).</li><li>Salazar, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Zinc+Transporter+ZnT3+interacts+with+AP-3+and+it+is+Targeted+to+a+Distinct+Synaptic+Vesicle+Subpopulation.">The Zinc Transporter ZnT3 interacts with AP-3 and it is Targeted to a Distinct Synaptic Vesicle Subpopulation.</a> Mol Biol Cell. 15, 575-587 (2004).</li><li>Trinkle-Mulcahy, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identifying+specific+protein+interaction+partners+using+quantitative+mass+spectrometry+and+bead+proteomes.">Identifying specific protein interaction partners using quantitative mass spectrometry and bead proteomes.</a> J Cell Biol. 183, 223-239 (2008).</li></ol>

DSP, a membrane-permeable, chemically reducible crosslinker with a spacer arm of 12 &Aring; is used to stabilize transient protein interactions 1,2,3,4. Here we exemplified this strategy with the adaptor complex AP-3 a soluble protein complex that recognizes and sorts membrane proteins into vesicles from endosomes 5. AP-3 selectively binds to the zinc transporter ZnT3 and the lipid kinase phosphatidylinositol-4-kinase type II alpha but not transferrin receptor 1,4,6. We expanded these obs...

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<html xmlns="http://www.w3.org/1999/xhtml" xml:lang="en" lang="en">	
		
		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Phosphate Buffered Saline</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>P4417</td>
<td>Dissolve 1 tablet in 200 mL water; add MgCl2 to a final concentration of 1 mM and CaCl2 to a final concentration of 0.1 mM</td>
</tr>
<tr>
<td>Dithiobis (succinimidyl propionate) (DSP)</td>
<td>&nbsp;</td>
<td>Thermo Scientific</td>
<td>22585</td>
<td>Moisture sensitive, store in air tight container 4&deg;C</td>
</tr>
<tr>
<td>Dimethyl sulphoxide (DMSO) Hybri-Max</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>D2650</td>
<td>&nbsp;</td>
<tr>
<td>Triton X-100, SigmaUltra</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>T9284</td>
<td>&nbsp;</td>
<tr>
<td>Dynabeads, Sheep anti-Mouse IgG</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>110.31</td>
<td>Beads are also available as sheep anti-rabbit</td>
</tr>
<tr>
<td>Dyna-Mag-2 magnet</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>123-21D</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

isolation of labile multi-protein complexes by in vivo controlled cellular cross-linking and immuno-magnetic affinity chromatography

The dynamic nature of cellular machineries is frequently built on transient and/or weak protein associations. These low affinity interactions preclude stringent methods for the isolation and identification of protein networks around a protein of interest. The use of chemical crosslinkers allows the selective stabilization of labile interactions, thus bypassing biochemical limitations for purification. Here we present a protocol amenable for cells in culture that uses a homobifunctional crosslinker with a spacer arm of 12 &Aring;, dithiobis-(succinimidyl proprionate) (DSP). DSP is cleaved by reduction of a disulphide bond present in the molecule. Cross-linking combined with immunoaffinity chromatography of proteins of interest with magnetic beads allows the isolation of protein complexes that otherwise would not withstand purification. This protocol is compatible with regular western blot techniques and it can be scaled up for protein identification by mass spectrometry1.
Stephanie A. Zlatic and Pearl V. Ryder contributed equally to this work.

Watch this Scientific Journal Video about Isolation of Labile Multi-protein Complexes by in vivo Controlled Cellular Cross-Linking and Immuno-magnetic Affinity Chromatography at JoVE.com

Isolation of Labile Multi-protein Complexes by in vivo Controlled Cellular Cross-Linking and Immuno-magnetic Affinity Chromatography

The cell permeable crosslinker DSP [dithiobis-(succinimidyl propionate)] stabilizes transient and labile interactions in vivo, which allows their isolation using stringent protein complex purification techniques. Here we present a technique for crosslinking cells grown in culture followed by isolation of protein complexes by immunoprecipitation.

Isolation of Labile Multi-protein Complexes by in vivo Controlled Cellular Cross-Linking and Immuno-magnetic Affinity Chromatography

The dynamic nature of cellular machineries is frequently built on transient and/or weak protein associations. These low affinity interactions preclude ...

isolation-labile-multi-protein-complexes-vivo-controlled-cellular

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JoVE Journal

Biology

17.3K 조회수.  Emory University. 세포 투과 crosslinker DSP [dithiobis - (succinimidyl 프로피 온산)] 일시적 안정화와 불안 정한 상호 작용 생체내에. 여기 immunoprecipitation에 의해 단백질 단지의 절연 다음 문화의 성장 crosslinking 전지에 대한 기술을 제시한다.

비디오: Isolation of Labile Multi-protein Complexes by in vivo Controlled Cellular Cross-Linking and Immuno-magnetic Affinity Chromatography

Photo-Induced Cross-Linking of Unmodified Proteins (PICUP) Applied to Amyloidogenic Peptides

The assembly of amyloidogenic proteins into toxic oligomers is a seminal event in the pathogenesis of protein misfolding diseases, including Alzheimer's, Parkinson's, and Huntington's diseases, hereditary amyotrophic lateral sclerosis, and type 2 diabetes. Owing to the metastable nature of these protein assemblies, it is difficult to assess their oligomer size distribution quantitatively using classical methods, such as electrophoresis, chromatography, fluorescence, or dynamic light scattering. Oligomers of amyloidogenic proteins exist as metastable mixtures, in which the oligomers dissociate into monomers and associate into larger assemblies simultaneously. PICUP stabilizes oligomer populations by covalent cross-linking and when combined with fractionation methods, such as sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) or size-exclusion chromatography (SEC), PICUP provides snapshots of the oligomer size distributions that existed before cross-linking. Hence, PICUP enables visualization and quantitative analysis of metastable protein populations and can be used to monitor assembly and decipher relationships between sequence modifications and oligomerization1. Mechanistically, PICUP involves photo-oxidation of Ru2+ in a tris(bipyridyl)Ru(II) complex (RuBpy) to Ru3+ by irradiation with visible light in the presence of an electron acceptor. Ru3+ is a strong one-electron oxidizer capable of abstracting an electron from a neighboring protein molecule, generating a protein radical1,2. Radicals are unstable, highly-reactive species and therefore disappear rapidly through a variety of intra- and intermolecular reactions. A radical may utilize the high energy of an unpaired electron to react with another protein monomer forming a dimeric radical, which subsequently loses a hydrogen atom and forms a stable, covalently-linked dimer. The dimer may then react further through a similar mechanism with monomers or other dimers to form higher-order oligomers. Advantages of PICUP relative to other photo- or chemical cross-linking methods3,4 include short (&#8804;1 s) exposure to non-destructive visible light, no need for pre facto modification of the native sequence, and zero-length covalent cross-linking. In addition, PICUP enables cross-linking of proteins within wide pH and temperature ranges, including physiologic parameters. Here, we demonstrate application of PICUP to cross-linking of three amyloidogenic proteins the 40- and 42-residue amyloid &beta;-protein variants (A&beta;40 and A&beta;42), and calcitonin, and a control protein, growth-hormone releasing factor (GRF).

Photo-Induced Cross-Linking of Unmodified Proteins PICUP Applied to Amyloidogenic Peptides

Photo-induced cross-linking of unmodified proteins (PICUP) allows characterization of oligomer size distribution in metastable protein mixtures. We demonstrate application of PICUP to three representative amyloidogenic peptides the 40- and 42-residue forms of amyloid &beta;-protein, and calcitonin, and a control peptide growth-hormone releasing factor.

The assembly of amyloidogenic proteins into toxic oligomers is a seminal event in the pathogenesis of protein misfolding diseases, including Alzheimer's, ...

Identification of protein complexes with quantitative proteomics in S. cerevisiae

Lipids are the building blocks of cellular membranes that function as barriers and in compartmentalization of cellular processes, and recently, as important intracellular signalling molecules. However, unlike proteins, lipids are small hydrophobic molecules that traffic primarily by poorly described nonvesicular routes, which are hypothesized to occur at membrane contact sites (MCSs). MCSs are regions where the endoplasmic reticulum (ER) makes direct physical contact with a partnering organelle, e.g., plasma membrane (PM). The ER portion of ER-PM MCSs is enriched in lipid-synthesizing enzymes, suggesting that lipid synthesis is directed to these sites and implying that MCSs are important for lipid traffic. Yeast is an ideal model to study ER-PM MCSs because of their abundance, with over 1000 contacts per cell, and their conserved nature in all eukaryotes. Uncovering the proteins that constitute MCSs is critical to understanding how lipids traffic is accomplished in cells, and how they act as signaling molecules. We have found that an ER called Scs2p localize to ER-PM MCSs and is important for their formation. We are focused on uncovering the molecular partners of Scs2p. Identification of protein complexes traditionally relies on first resolving purified protein samples by gel electrophoresis, followed by in-gel digestion of protein bands and analysis of peptides by mass spectrometry. This often limits the study to a small subset of proteins. Also, protein complexes are exposed to denaturing or non-physiological conditions during the procedure. To circumvent these problems, we have implemented a large-scale quantitative proteomics technique to extract unbiased and quantified data. We use stable isotope labeling with amino acids in cell culture (SILAC) to incorporate staple isotope nuclei in proteins in an untagged control strain. Equal volumes of tagged culture and untagged, SILAC-labeled culture are mixed together and lysed by grinding in liquid nitrogen. We then carry out an affinity purification procedure to pull down protein complexes. Finally, we precipitate the protein sample, which is ready for analysis by high-performance liquid chromatography/ tandem mass spectrometry. Most importantly, proteins in the control strain are labeled by the heavy isotope and will produce a mass/ charge shift that can be quantified against the unlabeled proteins in the bait strain. Therefore, contaminants, or unspecific binding can be easily eliminated. By using this approach, we have identified several novel proteins that localize to ER-PM MCSs. Here we present a detailed description of our approach. 

Here we describe a new quantitative proteomics technique for identifying protein complexes in Saccharomyces cerevisiae. In this study, we have used the SILAC method together with affinity purification followed by tandem mass spectrometry to identify with high specificity the binding partners of an ER protein, Scs2p.

Lipids are the building blocks of cellular membranes that function as barriers and in compartmentalization of cellular processes, and recently, as ...

Visualizing Single Molecular Complexes In Vivo Using Advanced Fluorescence Microscopy

Full insight into the mechanisms of living cells can be achieved only by investigating the key processes that elicit and direct events at a cellular level. To date the shear complexity of biological systems has caused precise single-molecule experimentation to be far too demanding, instead focusing on studies of single systems using relatively crude bulk ensemble-average measurements. However, many important processes occur in the living cell at the level of just one or a few molecules; ensemble measurements generally mask the stochastic and heterogeneous nature of these events. Here, using advanced optical microscopy and analytical image analysis tools we demonstrate how to monitor proteins within a single living bacterial cell to a precision of single molecules and how we can observe dynamics within molecular complexes in functioning biological machines. The techniques are directly relevant physiologically. They are minimally-perturbative and non-invasive to the biological sample under study and are fully attuned for investigations in living material, features not readily available to other single-molecule approaches of biophysics. In addition, the biological specimens studied all produce fluorescently-tagged protein at levels which are almost identical to the unmodified cell strains ("genomic encoding"), as opposed to the more common but less ideal approach for generating significantly more protein than would occur naturally ('plasmid expression'). Thus, the actual biological samples which will be investigated are significantly closer to the natural organisms, and therefore the observations more relevant to real physiological processes.

Visualizing Single Molecular Complexes In Vivo Using Advanced Fluorescence Microscopy

Here we demonstrate the protocols for performing single-molecule fluorescence microscopy on living bacterial cells to enable functional molecular complexes to be detected, tracked and quantified.

Full insight into the mechanisms of living cells can be achieved only by investigating the key processes that elicit and direct events at a cellular ...

ELIME (Enzyme Linked Immuno Magnetic Electrochemical) Method for Mycotoxin Detection

Immunoassays are a valid alternative to the more expensive and time consuming quantitative HPLC or GC1, 2 methods for the screening detection of hazardous mycotoxins in food commodities. In this protocol we show how to fabricate and interrogate an electrochemical competitive Enzyme linked immunomagnetic assay based on the use of magnetic beads as solid support for the immunochemical chain3 and screen printed electrodes as sensing platform.
Our method aims to determine the total amount of HT-2 and T-2 toxins, mycotoxins belonging to the trichothecenes family and of great concern for human health4. The use of an antibody clone with a cross reactivity of 100% towards HT-2 and T-2 allows to simultaneously detect both toxins with similar sensitivity5.
The first step of our assay is the coating step where we immobilize HT2-KLH conjugate toxin on the surface of magnetic beads. After a blocking step, necessary to avoid non-specific absorptions, the addition of a monoclonal antibody allows the competition between immobilized HT-2 and free HT-2 or T-2 present in the sample or dissolved in a standard solution.
At the end of the competition step, the amount of monoclonal antibody linked to the immobilized HT-2 will be inversely proportional to the amount of toxin in the sample solution.
A secondary antibody labeled with alkaline phosphatase (AP) is used to reveal the binding between the specific antibody and the immobilized HT-2. The final measurement step is performed by dropping an aliquot of magnetic bead suspension, corresponding to a specific sample/standard solution, on the surface of a screen-printed working electrode; magnetic beads are immobilized and concentrated by means of a magnet placed precisely under the screen-printed electrode. After two minutes of incubation between magnetic beads and a substrate for AP, the enzymatic product is detected by Differential Pulse Voltammetry (DPV) using a portable instrument (PalmSens) also able to initiate automatically eight measurements within an interval of few seconds.

ELIME Enzyme Linked Immuno Magnetic Electrochemical Method for Mycotoxin Detection

A protocol to detect trichothecenes (mycotoxins of concern for human health) using a newly developed screening method based on a competitive immunochemical method and a final electrochemical detection is demonstrated.

Immunoassays are a valid alternative to the more expensive and time consuming quantitative HPLC or GC1, 2 methods for the screening detection of hazardous ...

In vivo Imaging of Intact Drosophila Larvae at Sub-cellular Resolution

Recent improvements in optical imaging, genetically encoded fluorophores and genetic tools allowing efficient establishment of desired transgenic animal lines have enabled biological processes to be studied in the context of a living, and in some instances even behaving, organism. In this protocol we will describe how to anesthetize intact Drosophila larvae, using the volatile anesthetic desflurane, to follow the development and plasticity of synaptic populations at sub-cellular resolution1-3. While other useful methods to anesthetize Drosophila melanogaster larvae have been previously described4,5,6,7,8, the protocol presented herein demonstrates significant improvements due to the following combined key features: (1) A very high degree of anesthetization; even the heart beat is arrested allowing for lateral resolution of up to 150 nm1, (2) a high survival rate of &gt; 90% per anesthetization cycle, permitting the recording of more than five time-points over a period of hours to days2 and (3) a high sensitivity enabling us in 2 instances to study the dynamics of proteins expressed at physiological levels. In detail, we were able to visualize the postsynaptic glutamate receptor subunit GluR-IIA expressed via the endogenous promoter1 in stable transgenic lines and the exon trap line FasII-GFP1. (4) In contrast to other methods4,7 the larvae can be imaged not only alive, but also intact (i.e. non-dissected) allowing observation to occur over a number of days1. The accompanying video details the function of individual parts of the in vivo imaging chamber2,3, the correct mounting of the larvae, the anesthetization procedure, how to re-identify specific positions within a larva and the safe removal of the larvae from the imaging chamber.

In vivo Imaging of Intact Drosophila Larvae at Sub-cellular Resolution

This protocol describes a reliable method for anesthetization and imaging of intact Drosophila melanogaster larvae. We have utilized the volatile anesthetic desflurane to allow for repetitive imaging at sub-cellular resolution and re-identification of structures for up to a few days1.

Recent improvements in optical imaging, genetically encoded fluorophores and genetic tools allowing efficient establishment of desired transgenic animal ...

Isothermal Titration Calorimetry for Measuring Macromolecule-Ligand Affinity

Isothermal titration calorimetry (ITC) is a useful tool for understanding the complete thermodynamic picture of a binding reaction. In biological sciences, macromolecular interactions are essential in understanding the machinery of the cell. Experimental conditions, such as buffer and temperature, can be tailored to the particular binding system being studied. However, careful planning is needed since certain ligand and macromolecule concentration ranges are necessary to obtain useful data. Concentrations of the macromolecule and ligand need to be accurately determined for reliable results. Care also needs to be taken when preparing the samples as impurities can significantly affect the experiment. When ITC experiments, along with controls, are performed properly, useful binding information, such as the stoichiometry, affinity and enthalpy, are obtained. By running additional experiments under different buffer or temperature conditions, more detailed information can be obtained about the system. A protocol for the basic setup of an ITC experiment is given.

A general protocol for the use of isothermal titration calorimetry to monitor the binding thermodynamics for biological systems with moderate binding affinities is presented.

Isothermal titration calorimetry (ITC) is a useful tool for understanding the complete thermodynamic picture of a binding reaction.  In biological ...

Chromatin Isolation by RNA Purification (ChIRP)

Long noncoding RNAs are key regulators of chromatin states for important biological processes such as dosage compensation, imprinting, and developmental gene expression 1,2,3,4,5,6,7. The recent discovery of thousands of lncRNAs in association with specific chromatin modification complexes, such as Polycomb Repressive Complex 2 (PRC2) that mediates histone H3 lysine 27 trimethylation (H3K27me3), suggests broad roles for numerous lncRNAs in managing chromatin states in a gene-specific fashion 8,9. While some lncRNAs are thought to work in cis on neighboring genes, other lncRNAs work in trans to regulate distantly located genes. For instance, Drosophila lncRNAs roX1 and roX2 bind numerous regions on the X chromosome of male cells, and are critical for dosage compensation 10,11. However, the exact locations of their binding sites are not known at high resolution. Similarly, human lncRNA HOTAIR can affect PRC2 occupancy on hundreds of genes genome-wide 3,12,13, but how specificity is achieved is unclear. LncRNAs can also serve as modular scaffolds to recruit the assembly of multiple protein complexes. The classic trans-acting RNA scaffold is the TERC RNA that serves as the template and scaffold for the telomerase complex 14; HOTAIR can also serve as a scaffold for PRC2 and a H3K4 demethylase complex 13.
Prior studies mapping RNA occupancy at chromatin have revealed substantial insights 15,16, but only at a single gene locus at a time. The occupancy sites of most lncRNAs are not known, and the roles of lncRNAs in chromatin regulation have been mostly inferred from the indirect effects of lncRNA perturbation. Just as chromatin immunoprecipitation followed by microarray or deep sequencing (ChIP-chip or ChIP-seq, respectively) has greatly improved our understanding of protein-DNA interactions on a genomic scale, here we illustrate a recently published strategy to map long RNA occupancy genome-wide at high resolution 17. This method, Chromatin Isolation by RNA Purification (ChIRP) (Figure 1), is based on affinity capture of target lncRNA:chromatin complex by tiling antisense-oligos, which then generates a map of genomic binding sites at a resolution of several hundred bases with high sensitivity and low background. ChIRP is applicable to many lncRNAs because the design of affinity-probes is straightforward given the RNA sequence and requires no knowledge of the RNA's structure or functional domains.

Chromatin Isolation by RNA Purification ChIRP

ChIRP is a novel and rapid technique to map genomic binding sites of long noncoding RNAs (lncRNAs). The method takes advantage of the specificity of anti-sense tiling oligonucleotides to allow the enumeration of lncRNA-bound genomic sites.

Long noncoding RNAs are key regulators of chromatin states for important biological processes such as dosage compensation, imprinting, and developmental ...

Identification of Protein Complexes in Escherichia coli using Sequential Peptide Affinity Purification in Combination with Tandem Mass Spectrometry

Since most cellular processes are mediated by macromolecular assemblies, the systematic identification of protein-protein interactions (PPI) and the identification of the subunit composition of multi-protein complexes can provide insight into gene function and enhance understanding of biological systems1, 2. Physical interactions can be mapped with high confidence vialarge-scale isolation and characterization of endogenous protein complexes under near-physiological conditions based on affinity purification of chromosomally-tagged proteins in combination with mass spectrometry (APMS). This approach has been successfully applied in evolutionarily diverse organisms, including yeast, flies, worms, mammalian cells, and bacteria1-6. In particular, we have generated a carboxy-terminal Sequential Peptide Affinity (SPA) dual tagging system for affinity-purifying native protein complexes from cultured gram-negative Escherichia coli, using genetically-tractable host laboratory strains that are well-suited for genome-wide investigations of the fundamental biology and conserved processes of prokaryotes1, 2, 7. Our SPA-tagging system is analogous to the tandem affinity purification method developed originally for yeast8, 9, and consists of a calmodulin binding peptide (CBP) followed by the cleavage site for the highly specific tobacco etch virus (TEV) protease and three copies of the FLAG epitope (3X FLAG), allowing for two consecutive rounds of affinity enrichment. After cassette amplification, sequence-specific linear PCR products encoding the SPA-tag and a selectable marker are integrated and expressed in frame as carboxy-terminal fusions in a DY330 background that is induced to transiently express a highly efficient heterologous bacteriophage lambda recombination system10. Subsequent dual-step purification using calmodulin and anti-FLAG affinity beads enables the highly selective and efficient recovery of even low abundance protein complexes from large-scale cultures. Tandem mass spectrometry is then used to identify the stably co-purifying proteins with high sensitivity (low nanogram detection limits).
Here, we describe detailed step-by-step procedures we commonly use for systematic protein tagging, purification and mass spectrometry-based analysis of soluble protein complexes from E. coli, which can be scaled up and potentially tailored to other bacterial species, including certain opportunistic pathogens that are amenable to recombineering. The resulting physical interactions can often reveal interesting unexpected components and connections suggesting novel mechanistic links. Integration of the PPI data with alternate molecular association data such as genetic (gene-gene) interactions and genomic-context (GC) predictions can facilitate elucidation of the global molecular organization of multi-protein complexes within biological pathways. The networks generated for E. coli can be used to gain insight into the functional architecture of orthologous gene products in other microbes for which functional annotations are currently lacking.

Identification of Protein Complexes in Escherichia coli using Sequential Peptide Affinity Purification in Combination with Tandem Mass Spectrometry

Affinity purification of tagged proteins in combination with mass spectrometry (APMS) is a powerful method for the systematic mapping of protein interaction networks and for investigating the mechanistic basis of biological processes. Here, we describe an optimized sequential peptide affinity (SPA) APMS procedure developed for the bacterium Escherichia coli that can be used to isolate and characterize stable multi-protein complexes to near homogeneity even starting from low copy numbers per cell.

Since most cellular processes are mediated by macromolecular assemblies, the systematic identification of protein-protein interactions (PPI) and the ...

AFM-based Mapping of the Elastic Properties of Cell Walls: at Tissue, Cellular, and Subcellular Resolutions

We describe a recently developed method to measure mechanical properties of the surfaces of plant tissues using atomic force microscopy (AFM) micro/nano-indentations, for a JPK&#160;AFM. Specifically, in this protocol we measure the apparent Young&#8217;s modulus of cell walls at subcellular resolutions across regions of up to 100 &#181;m&#160;x 100 &#181;m in floral meristems, hypocotyls, and roots. This requires careful preparation of the sample, the correct selection of micro-indenters and indentation depths. To account for cell wall properties only, measurements are performed in highly concentrated solutions of mannitol in order to plasmolyze the cells and thus remove the contribution of cell turgor pressure.
In contrast to other extant techniques, by using different indenters and indentation depths, this method allows simultaneous multiscale measurements, i.e. at subcellular resolutions and across hundreds of cells comprising a tissue. This means that it is now possible&#160;to spatially-temporally characterize the changes that take place in the mechanical properties of cell walls during development, enabling these changes to be correlated with growth and differentiation. This represents a key step to understand how coordinated microscopic cellular changes bring about macroscopic morphogenetic events.
However, several limitations remain: the method can only be used on fairly small samples (around 100 &#181;m in diameter) and only on external tissues; the method is sensitive to tissue topography; it measures only certain aspects of the tissue&#8217;s complex mechanical properties. The technique is being developed rapidly and it is likely that most of these limitations will be resolved in the near future.

We describe a method to map mechanical properties of plant tissues using an atomic force microscope (AFM). We focus on how to record mechanical changes that take place in cell walls during plant development at wide-field mesoscale, enabling these changes to be correlated with growth and morphogenesis.

We describe a recently developed method to measure mechanical properties of the surfaces of plant tissues using atomic force microscopy (AFM) ...

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) maintain the turnover of all mature blood cell lineages. The coordination of the complex signals leading to specific HSC fates relies upon the interaction between HSCs and the intricate bone marrow microenvironment, which is still poorly understood[1-2].
We describe how by combining a newly developed specimen holder for stable animal positioning with multi-step confocal and two-photon in vivo imaging techniques, it is possible to obtain high-resolution 3D stacks containing HSPCs and their surrounding niches and to monitor them over time through multi-point time-lapse imaging. High definition imaging allows detecting ex vivo labeled hematopoietic stem and progenitor cells (HSPCs) residing within the bone marrow. Moreover, multi-point time-lapse 3D imaging, obtained with faster acquisition settings, provides accurate information about HSPC movement and the reciprocal interactions between HSPCs and stroma cells.
Tracking of HSPCs in relation to GFP positive osteoblastic cells is shown as an exemplary application of this method. This technique can be utilized to track any appropriately labeled hematopoietic or stromal cell of interest within the mouse calvarium bone marrow space.

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

The nature of the interactions between hematopoietic stem and progenitor cells (HSPCs) and bone marrow niches is poorly understood. Custom hardware modifications and a multi-step acquisition protocol allow the use of two-photon and confocal microscopy to image ex vivo labeled HSPCs homed within bone marrow areas, tracking interactions and movement.

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) ...